Fiver portions of about 3 mL of water was added in increments and cooled in an ice bath for about 10 minutes. After a new filter paper was add and the aspirin was placed in while the vacuum filtration was on and about 6 mL was poured in to get the last amount of
Equal volume of chloroform was added and centrifuged at 13000 rpm for 5 minutes. Again, the aqueous solution was transferred to a new 1.5 ml microcentrifuged tube and 1/10 volume of 3M Sodium Acetate Solution was added. Then, 2.5 volumes of cold absoluted ethanol were added to precipitate the DNA. The mixture was incubated in -20 °C for overnight or -80 °C for 1-2 hour. After incubation, the mixture was then spinning at 13000 rpm for 30 minutes at 4 °C.
The phthalimide will dissolve within 5 minutes. Place in the freezer again for at least 30 minutes. Now add the cold NaOH solution prepared above to the cold (not over +5°C) Alclorite solution. Allow to stand at room temperature until temperature reaches 20°C. Now heat to 80-85°C over 10 minutes and hold there for at least two minutes but not more than five.
Then add two drops of iodine reagent to each of these wells and wait one minute, then add 0.5ml of amylase to each of the appropriate tubes. After two minutes, add a few drops from each tube, and place in the corresponding wall. After collecting the data use a color coding scheme to convert the qualitative data to quantitative data. The numerical data from each group was used to calculate the mean starch concentration and the standard deviation. The mean and the standard deviation values were calculated after entering all the data into
Protein Extraction and Purification. The USP7 bacterial pellet was thawed at RT, 1 mL of cold Ni 2+ Lysis/Binding Buffer (5 mL of Common Buffer + 5 uL of 5 M Imidazole, which had an Imidazole concentration of 5 mM) was added to the bacterial pellet and thoroughly vortexed. The cells were lysed using the sonicator (30% intensity, 4 x 20 sec pulses, and 15 sec gaps). Unbroken cells and insoluble proteins were sediment using the refrigerated micro-centrifuge (... ... middle of paper ... ...ed and further studies will be necessary to determine what the interaction affects or how the two proteins interact. References Department of Biology 2013, BIOL 2070 Research Methods in Cell and Molecular Biology, Resource Manual, pp 98-124.
Next, the beads were washed three times with cell lysis buffer, and boiled with 50 μL of 1xSDS sample buffer at 95℃ for 5 min. After centrifuging the beads at 13, 000rpm for 5 min, 10 μL of eluted Anxa2 interacting proteins were initially separated by SDS-PAGE and analyzed by Commassie Blue Staining, and the other eluted proteins were further analyzed by mass spectrometric analysis or western blotting analysis.
Third, the remaining serum free medium and complete growth medium plates were analyzed. This assay was carried out by replacing the medium with 0.5 ml yellow MTT solution (Sigma) and incubated for two hours. Then, the MTT solution was replaced with 0.6 ml of MTT solvent (isopropanol) (BDH) and the plates were placed on a gyratory shaker for 15 minutes. Each well was split in triplicate in a 96 well plate in equal volumes. The absorbance was measured using plate reader at a wavelength of 570 nm.
Centrifugation The microfuge tube was set aside on ice while the remaining filtrate was centrifuged at 200g’s. The supernatant was removed and 100ul were set aside in a tube labeled “LSS” (for low speed supernatant) on ice. The remaining supernatant was centrifuged at 1000g’s for 7 minutes. 100 uL were taken from supernatant, and placed in a microfuge tube on ice labeled “HSS.” The pellet was resuspended in 250 ul of homogenization buffer. 100 uL of this solution was set aside in a tube labeled “HSS.” SDS-PAGE preparation.
All the six HER2 ECD-expressing clones showed the biotinylation of HER2 ECD in ELISA assay (Fig. 2-B). The medium of one selected clonal cells cultured in the presence of biotin, were dialyzed extensively against phosphate buffer to eliminate free biotin an... ... middle of paper ... ...tal efficacy . Glycosylphosphatidylinositol (GPI) anchors play important roles in the surface expression of eukaryotic membrane proteins . GPI precursor is synthesized in the endoplasmic reticulum and then is covalently attached as a posttranslational modification to the C-terminal of a protein that express a GPI attachment signal sequence .
Then 25.0ml of hexane was added and the mixture was shaken for 5 minutes with occasional venting. The aqueous layer was discarded and the organic layer was left inside. About 25.0ml of 10% NaOH was then added and the mixture was shaken as before. The aqueous layer was collected and then cooled in an ice bath. It was then acidified with enough 6.00 M HCl while the pH is being monitored with red litmus paper.